Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients’ plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.

Administration of Recombinant Soluble Urokinase Receptor Per Se Is Not Sufficient to Induce Podocyte Alterations and Proteinuria in Mice

“…29 Accordingly, uPAR is absent from the cell surface of leukocytes affected by the PNH driver mutations; these abnormal cells respond by secreting a soluble intact receptor, thus sustaining chronically elevated levels of suPAR in the plasma of patients with PNH. [29][30][31][32] Despite these abnormally high levels of suPAR in the plasma of patients with PNH, to the best of our knowledge, FSGS is not one of the prevalent renal lesions observed in these patients. 33 Renal dysfunction does occur in patients with PNH, but most of these clinical features can be alleviated by eculizumab treatment, highlighting chronic complement-mediated hemolysis as the driver of renal pathogenesis.…”

Loss of expression of neutrophil proteinase-3: a factor contributing to thrombotic risk in paroxysmal nocturnal hemoglobinuria

“…A recent study showed that factors correlating with markedly elevated levels of soluble uPAR included the absolute number and percent of GPI-negative neutrophils, and absolute neutrophil count. 20 Other investigators highlighted enhanced platelet activation and elevated levels of circulating procoagulant microparticles derived from platelets and/or endothelial cells. [12][13][14][15] It has also been suggested that PNH platelets, which likely correlate with the size of PNH clone, may trigger a prothombotic cascade due to lack of certain GPI-deficient proteins.…”

“…Previous studies investigating thrombophilia in PNH implicated several possible mechanisms; however, none of the pathways described to date provide sufficient explanation for the extraordinarily high risk of thrombosis in these patients. [11][12][13][14][15][16][17][18][19][20] For example, abnormalities of the fibrinolytic cascade mediated through deficiency of the GPIlinked uPAR normally present on platelets and leukocytes have been described. Competitive binding of pro-uPA by an excess of soluble uPAR could contribute to a deficiency of fibrinolytic activity as could the loss of active uPA normally localized to thrombi by binding to uPAR on cells within a clot.…”

“…10 While the pathogenesis of the thrombophilia in PNH has not been clarified, a number of potential mechanisms have been proposed, including episodic hemolysis with release of pro-coagulant microparticles, [11][12][13] complementmediated platelet activation, 9,12,14,15 and defective fibrinolytic activity secondary to loss of leukocyte expression of the GPI-linked urokinase-type plasminogen activator receptor (uPAR). [16][17][18][19][20] However, none of these hypotheses alone adequately explains the marked degree of hemostatic activation that results in a strikingly higher incidence of thromboembolic complications in PNH.…”

Experimental concerns regarding suPAR-related proteinuria

“…This antibody recognizes the human β3 integrin GPINCT sequence, which is absent in mouse β3 integrin 8 . Third, paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired bone marrow disorder in which suPAR overproduc tion and/or accumulation is linked to the dysfunction of glycosylphosphatidylinositol anchoring, leading to very high levels of circulating suPAR [9][10][11] . As the disease occurs in haematopoietic stem cells (at least in early myeloid progenitor cells), PNH might represent the human counterpart of experimental conditions discussed in the commentary (that is, the effects of high levels of circulating suPAR coming from haematopoietic sources).…”